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Sequence Results

Sequence Results. T he A rabidopsis I nformation R esource. The National Center for Genome Resources (NCGR), Santa Fe, New Mexico The Arabidopsis Biological Resource Center (ABRC), Ohio State University Carnegie Institution, Department of Plant Biology, Stanford University .

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Sequence Results

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  1. Sequence Results

  2. The Arabidopsis Information Resource The National Center for Genome Resources (NCGR), Santa Fe, New Mexico The Arabidopsis Biological Resource Center (ABRC), Ohio State University Carnegie Institution, Department of Plant Biology, Stanford University Mission Summary: Provide research resources for the Arabidopsis research community

  3. NSF 2010 Project To determine the function of all genes in Arabidopsis thaliana by the year 2010

  4. How To Determine Gene Function? • Gene Overview • Knockouts • Localization

  5. Getting an overview of your gene.

  6. www.arabidopsis.org

  7. P450s are membrane-associated proteins, either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells where they metabolize thousands of endogenous and exogenous compounds.

  8. What does the number At1g11680 mean? At (Arabidopsis thaliana) 1,2,3,4,5 (chromosome number) or M for mitochondrial or C for chloroplast. g (gene), other letters possible for repeats etc.) 12300 (five-digit code, numbered from top/north to bottom/south of chromosome)

  9. Publications describing your gene.

  10. Obtaining a knockout of gene At1g11680

  11. SIGnAL Salk Institute Genomic Analysis Laboratory

  12. Frequently asked questions answers a lot of questions!!!

  13. Localizing expression of gene At1g11680

  14. Cre-loxP Site-Specific Recombination (cyclization recombination)

  15. Cre-LoxP for Promoter GUS fusion

  16. Cre recombinase (Cre): A type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites The loxP recognition element:

  17. pUNI pHOST 10x BufferWaterCre 1µl1µl2µl15µl1µl 37°C Transform in DH5α.Select on Amp. 20 min 20µl Qiagen miniprep DNA, approx. 200 ng/µl Recommended reaction for Cre-LoxP

  18. Univector Plasmid-Fusion System

  19. β-Glucuronidase Green Fluorescent Protein

  20. Luciferase

  21. β-Glucuronidase General localization. X-Gluc + β-Glucuronidase = Blue

  22. Green Fluorescent Protein General localization and subcellular localization.

  23. Luciferase Evaluation of gene expression/induction.

  24. In Summary: The Arabidopsis Information Resource • The primary goal of TAIR is to assist the Arabidopsis community discover gene function. • Extensive gene knockout and localization tools (T-DNA lines, full-length cDNAs, and pUNI cDNAs) serve as the core elements of what TAIR offers. • The TAIR website, its databases, and resources continue to expand, so if you are unable to find a T-DNA insertion today…try tomorrow! Syngenta Inc has now donated about 9,000 more lines to ABRC and sequences to this site at Mar. 31 2006

  25. Key References Cre-loxP recombination vectors for the expression of Riken Arabidopsis full-length cDNAs in plants Toshiro Shigaki, Mamata Kole, John M. Ward, Heven Sze, and Kendal D. Hirschi BioTechniques Vol. 39, No. 3: pp 301-303 (Sep 2005) The Cre-loxP recombination-based reporter system for plant transcriptional expression studies Shigaki, T., R. Ravindranadha, A.B. Sivitz, J.M. Ward, H. Sze, and K.D. Hirschi Plant Mol. Biol. 58:65-73 (2005) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes Liu, Q., M.Z. Li, D. Leibham, D. Cortez, and S.J. Elledge Curr. Biol. 8:1300-1309 (1998) A structural view of Cre-loxP site-specific recombination Gregory D. Van Duyne Annual Review of Biophysics and Biomolecular Structure Vol. 30: 87-104 (2001)

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